Acyl-chain selectivity and physiological roles ofStaphylococcus aureus fatty acid– binding proteinsReceived for publication, October 5, 2018, and in revised form, October 25, 2018 Published, Papers in Press, November 14, 2018, DOI 10.1074/jbc.RA118.006160

Maxime G. Cuypers‡, X Chitra Subramanian§, Jessica M. Gullett§, Matthew W. Frank§, Stephen W. White‡,and X Charles O. Rock§1

From the Departments of ‡Structural Biology and §Infectious Diseases, St. Jude Children’s Research Hospital,Memphis, Tennessee 38105

Edited by George M. Carman

Fatty acid (FA) kinase produces acyl-phosphate for the syn-thesis of membrane phospholipids in Gram-positive bacterialpathogens. FA kinase consists of a kinase protein (FakA) thatphosphorylates an FA substrate bound to a second module, anFA-binding protein (FakB). Staphylococcus aureus expressestwo distinct, but related, FakBs with different FA selectivities.Here, we report the structures of FakB1 bound to four saturatedFAs at 1.6 –1.93 Å resolution. We observed that the different FAstructures are accommodated within a slightly curved hydro-phobic cavity whose length is governed by the conformation ofan isoleucine side chain at the end of the tunnel. The hydropho-bic tunnel in FakB1 prevents the binding of cis-unsaturated FAs,which are instead accommodated by the kinked tunnel withinthe FakB2 protein. The differences in the FakB interiors are notpropagated to the proteins’ surfaces, preserving the protein–protein interactions with their three common partners, FakA,PlsX, and PlsY. Using cellular thermal shift analyses, we foundthat FakB1 binds FA in vivo, whereas a significant proportion ofFakB2 does not. Incorporation of exogenous FA into phospho-lipid in �fakB1 and �fakB2 S. aureus knockout strains revealedthat FakB1 does not efficiently activate unsaturated FAs. FakB2preferred unsaturated FAs, but also allowed the incorporationof saturated FAs. These results are consistent with a model inwhich FakB1 primarily functions in the recycling of the satu-rated FAs produced by S. aureus metabolism, whereas FakB2activates host-derived oleate, which S. aureus does not producebut is abundant at infection sites.

Fatty acid (FA)2 kinase of Gram-positive bacteria is respon-sible for the activation of FA prior to their incorporation into

membrane phospholipids. The enzyme system consists of akinase module (FakA) that binds to a FA-binding protein(FakB) and phosphorylates FakB(FA) to create an acyl-PO4(FakB(FA�P)) (1, 2). The FakB(FA�P) is either utilized by theglycerol-phosphate acyltransferase (PlsY) to initiate phospho-lipid synthesis or is converted to acyl-acyl carrier protein (ACP)by acyl-phosphate:ACP transacylase (PlsX) to enter the elonga-tion cycle of bacterial FA biosynthesis. FA kinase is the onlypathway for FA activation and incorporation into Staphylococ-cus aureus membrane phospholipids and has an important rolein activating exogenous FA, but it is also involved in cellularlipid homeostasis (3). FA are generated by metabolism inS. aureus, and in the absence of kinase activity, the levels ofintracellular FA rise (3). FA kinase–null S. aureus are resistantto dermcidin (4) and exhibit increased biofilm formation (5).However, the most striking phenotype of FA kinase knockoutstrains is the lack of �-hemolysin production indicating a novelrole for FA kinase in controlling the production of virulencefactors (6). A genome-wide analysis shows FA kinase-nullstrains are deficient in virulence factor transcription controlledby the SaeRS two-component system (2). The absence of FAkinase leads to a severe depression in the transcription of viru-lence factor genes due to the inhibitory effect of cellular FA onSaeRS signaling (3). The structure of S. aureus FakB2 bound tooleic acid (18:1) is a model for the functional importance of thehighly conserved residues in the FakB protein family (7). Theacyl chain of FakB2(18:1) is completely buried in the proteininterior with only the carboxyl group exposed at the proteinsurface. Ser-93, Thr-61, and His-266 form a conserved hydro-gen bond network that fixes the position of the FA carboxylmoiety. In addition, Arg-202 is a conserved surface residue thatis required for FakB2 to interact optimally with FakA (7).

S. aureus has two FakB proteins: FakB1 that binds saturatedFA and FakB2 that is specific for monounsaturated FA (2).Other bacteria have between one and four FakB homologs, butthe physiological roles of multiple FA-binding proteins and thestructural basis for acyl chain selectivity remain open questions.Here, we report the structures of S. aureus FakB1 bound to fourdifferent straight and branched-chain saturated FA produced

This work was supported by National Institutes of Health Grant GM034496,Cancer Center Support Grant CA21765, and the American Lebanese Asso-ciated Charities. The authors declare that they have no conflicts of interestwith the contents of this article. The content is solely the responsibility ofthe authors and does not necessarily represent the official views of theNational Institutes of Health.

This article was selected as one of our Editors’ Picks.The atomic coordinates and structure factors (codes 5UXY, 5V85, 5WOO,

6ALW, 5UTO, and 6B9I) have been deposited in the Protein Data Bank(http://wwpdb.org/).

1 To whom correspondence should be addressed: Dept. of Infectious Dis-eases, St. Jude Children’s Research Hospital, 262 Danny Thomas Place,Memphis, TN 38105. Tel.: 901-595-3491; E-mail: charles.rock@stjude.org.

2 The abbreviations used are: FA, fatty acid; FA�P, acyl-phosphate; [D4]16:0,tetradeuteriohexadecanoic acid; ACP, acyl carrier protein; BSA, bovineserum albumin; LPA, 1-acyl-sn-glycerol-3-phosphate; PlsX, acyl-phos-

phate:ACP transacylase; PlsY, acyl-phosphate-dependent glycerol-phos-phate acyltransferase; 15:0, FA designated by number of carbons:numberof double bonds; a, anteiso; PG, phosphatidylglycerol; PDB, Protein DataBank; CETSA, cellular thermal shift assay; bis-tris, 2-[bis(2-hydroxyethyl)-amino]-2-(hydroxymethyl)propane-1,3-diol; SFA, saturated fatty acid; PEG,polyethylene glycol.

croEDITORS’ PICK

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by the bacterium. FakB1 and FakB2 have acyl chain bindingtunnels with distinctly different shapes that account for the acylchain selectivity of the two proteins, but the protein surfaces areconserved. This allows the two FakBs to not only interact withFakA, but also with the two downstream enzymes that utilizeFakB(FA�P) as substrates in bacterial lipid metabolism (PlsXand PlsY). Cellular thermal shift experiments performed withgrowing S. aureus suggest that FakB1 normally has a bound FA,whereas FakB2 does not. These data lead to a model whereFakB1 functions in cellular lipid homeostasis in the absence ofexogenous FA, whereas the key function of FakB2 is to activateenvironmental 18:1�9, an abundant host FA that S. aureus doesnot produce but is incorporated into membrane phospholipidsby the FA kinase pathway.

Results

FakB1 structure

A single FakB1-FA species for crystallization trials wasobtained by exchanging 16:0 into purified FakB1 and removingexcess FA by gel-filtration chromatography (7). FakB1(16:0)crystallized in space group P1 with two monomers in theasymmetric unit, and the crystal diffracted to 1.83 Å. TheFakB1(16:0) structure shows an overall two-domain proteinfold very similar to those of other FakB protein family members(Fig. 1A and Table 1) (7–9). The N-terminal domain consistsof an EDD fold (10) with an extension comprising a three-stranded anti-parallel �-sheet and one �-helix. The C-terminal

domain contains a six-stranded �-sheet flanked by two �-heli-ces on one side and three on the other side (Fig. 1A). The struc-tures of FakB1(16:0) and the previously determined FakB2(18:1)superimpose very well (Fig. 1A). Arg-205 in FakB1 is in theequivalent position to Arg-202 in FakB2 that is required forhigh-affinity binding to FakA (7) and presumably mediates thesame interaction in FakB1. An exposed flexible loop adjacent tothe FA carboxyl group was not visible in the FakB2(18:1) struc-ture (residues 174 –183), but this loop in FakB1 (16:0) (residues176 –187) is resolved in one of the two monomers of the asym-metric unit (monomer A) (Fig. 1A, Loop). This loop is adjacentto Arg-205 and may also be involved in the interaction withFakA, although its primary sequence is not conserved whencompared with the FakB2 loop.

An overlay of the FakB1(16:0) and FakB2(18:1) structurescentered on the FA carboxyl group reveals that the carboxyl-binding sites are essentially identical (Fig. 1B). The carboxylgroup is held firmly in place by a hydrogen bond network con-sisting of a serine on one side and a threonine– histidine pair onthe other side. The four FakB1 residues, Thr-62, Ser-95, His-270, and Arg-173, correspond to Thr-61, Ser-93, His-266, andArg-170 in FakB2. Arg-170 is essential for catalysis in FakB2 (7),and the proximal apolar region of the side chain packs along theacyl chain of the bound FA. Arg-173 mediates the same inter-action with the bound FA in FakB1. As noted for FakB2, thelocation of Arg-173 suggests that it is ideally positioned to interactwith the dianionic FA�P product bound to FakB1 and/or to par-

Figure 1. FakB1(16:0) structure. A, structures of FakB1(16:0) (light gray) and FakB2(18:1�9) (rainbow) are overlaid. The color of the FakB2 structure is given bythe structural alignment distance between the FakB1 and FakB2 main chain C� atoms, illustrating the structural similarities and dissimilarities from blue to red,respectively. The main chain C� atom distance color scale is presented at bottom left. Fatty acid colors match their respective protein and are presented in balland stick. The locations of Arg-202 (FakB2) and Arg-205 (FakB1) are conserved. Loop is observed in FakB1, but not FakB2. B, overlay of the fatty acid carboxylgroups from FakB1(16:0) in white and FakB2(18:1�9) in blue. The FA-only calculated alignment was performed with PSICO/MCSALIGN plugin for PyMOL version1.8 (Schrödinger, LLC). The tunnel residues are presented as semitransparent sticks only and the fatty acids as ball and sticks. Water molecules are single coloredspheres. Hydrogen bond networks are presented as dotted lines between atoms. C, positioning of Arg-173 and the hydrogen bond network that connects theguanidinium group to a constellation of structured water molecules and the fatty acid carboxyl group.

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ticipate in the phosphotransfer reaction (Fig. 1C). Arg-173 is con-nected to a network of three water molecules that hovers above thecarboxyl group (Fig. 1C), and one can envision that the phosphateoxygens would displace these water molecules in a phosphorylatedFakB1(16:0-PO4) intermediate.

FakB1 complexes with 14:0, a15:0, and a17:0 FA

Multiple saturated straight and branched-chain FA arefound in S. aureus phospholipids, which suggests that theFakB1 hydrophobic acyl chain binding tunnel should be flexibleenough to accommodate this spectrum of FA. The structures ofFakB1 in complex with 14:0, a15:0, and a17:0 FA were obtainedby exchanging the indicated FA into FakB1, and re-purifyingthe protein complexes by gel-filtration chromatography toremove unbound FA and detergent prior to crystallization (7).The electron densities of all four of the FA in the FakB1 struc-tures are illustrated in Fig. 2A. The FakB1 tunnel is a slightlycurved space that expands and contracts to accommodate thedifferent FA chain lengths. This movement arises at the termi-nus of the FakB1 FA binding tunnel through the re-positioningof the Ile-233 side chain that functions like a “swinging gate”that undergoes a hinge-like motion to either increase ordecrease the length of the FA-binding channel (Fig. 2, B and C).This movement of Ile-233 allows the tunnel to easily accommo-date the two additional carbons in the FakB1(16:0) structure com-

pared with the FakB1(14:0) structure. Branched-chain anteiso-FA(a15:0 and a17:0) are major constituents of S. aureus phospholip-ids, and they are exclusively the S stereoisomer (11). When a race-mic mixture (S and R) of a15:0 was exchanged into FakB1 prior tocrystallization, both stereoisomers were represented in the elec-tron density map, although there was a preference for the S isomer(60% S and 40% R). In contrast, only the S isomer was observed inthe FakB1 tunnel when the experiment was performed using race-mic a17:0. Thus, the additional carbon atoms in a17:0 clearlyimpose a stricter chain-length and stereospecificity within theFakB1 tunnel. These data show that the binding tunnel in FakB1is structurally tuned to accommodate the major straight andbranched-chain FA produced by S. aureus.

There are many FakB structures in the PDB database, but notall are refined with FA in the acyl-chain binding tunnel. This isdue in part to the structures being determined prior to the dis-covery that they are FA-binding proteins in the FA kinase sys-tem and that FakBs may bind multiple FA. If care is not taken toexchange a single FA into the pocket, the electron density willreflect the mixture of FA structures that were picked up by theprotein during the protein expression. Purified FakBs musthave a ligand bound in the pocket to stabilize the protein andallow it to be manipulated in vitro. We examined and re-refinedtwo deposited FakB structures, from Ruminococcus gnavus (old

Table 1Data collection statistics for FakB1–fatty acid complexesThe abbreviations are: myristic acid (14:0), 12-methyltetradecanoic acid (a15:0), palmitic acid (16:0), and 14-methylhexadecanoic acid (a17:0) are shown. Values inparentheses are for the highest resolution shell. ML is maximum likelihood. Rmerge � �(I � �I�)/�(I), where I is the intensity measured for a given reflection, and �I� is theaverage intensity for multiple measurements of this reflection. Rp.i.m. � (�[1/(N � 1)]∧1/2. ��I � �I��)/�(I). Rwork � ��Fobs� � �Fcalc�/��Fobs�; where Fobs and Fcalc are theobserved and calculated structure factor amplitudes, respectively. Rfree is Rwork for the 5% excluded reflections during refinement.

Fatty acids 14:0 a15:0 16:0 a17:0

PDB codes 5WOO 6ALW 5UTO 6B9IData collection

Beamline SER-CAT 22-ID SER-CAT 22-BM SER-CAT 22-ID SER-CAT 22-BMTemperature (K) 100 100 100 100Wavelength (Å) 1.0000 1.0000 1.0000 1.0000Space group P1 P1 P1 P1Unit cell parameters (Å)

a, b, c 33.39, 54.45, 84.91 33.35, 54.53, 84.42 33.19, 54.29, 84.40 32.99, 54.17, 83.90�, �, � 104.89, 90.21, 107.76 105.13, 90.00, 107.81 105.18, 90.02, 107.50 104.71, 90.00, 107.73

Resolution range (Å) 81.73–1.78 81.18–1.63 48.83–1.83 80.87–1.93Rmerge 0.086 (0.676) 0.184 (0.906) 0.067 (0.641) 0.135 (0.629)Rp.i.m. 0.051 (0.395) 0.106 (0.524) 0.046 (0.443) 0.080 (0.592)No. of observations 194,540 (11,418) 263,005 (13,084) 141,973 (8479) 150,843 (5254)No. of unique reflections 50,586 (2913) 66,112 (3294) 46,256 (2815) 39,271 (2578)Multiplicity 3.8 (3.9) 4.0 (4.0) 3.1 (3.0) 3.8 (2.0)Mean I/�(I) 9.8 (2.0) 8.6 (2.0) 12.3 (1.9) 10.1 (1.9)Mean (I) half-set correlation 0.996 (0.736) 0.985 (0.662) 0.998 (0.694) 0.992 (0.487)Completeness (%) 96.2 (95.9) 97.2 (95.2) 97.1 (95.3) 97.9 (96.4)Wilson B-factor (Å�2) 19.68 7.76 15.21 16.73

Model qualityRwork/Rfree value (%) 15.1/20.0 15.4/19.6 15.8/19.1 16.6/20.6Root mean square deviations

Bond lengths (Å) 0.006 0.010 0.003 0.003Bond angles (°) 0.801 0.961 0.540 0.550

Coordinates error (ML, Å) 0.17 0.18 0.19 0.21Protein residues 575 569 575 561Average B-factor (Å�2)

All atoms 27.54 16.46 25.95 22.63Protein atoms 25.65 13.58 24.62 21.43Fatty acid atoms 24.15 24.23 15.68 30.86Solvent atoms 40.97 31.24 36.11 32.79

Ramachandran plotFavored (%) 98.59 98.22 97.36 98.92Allowed (%) 1.23 1.42 1.76 1.08Outliers (%) 0.18 0.36 0.88 0.00

Clashscore 6.29 6.21 3.42 2.64

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PDB code 3JR7) and Eubacterium eligens (old PDB code 3FDJ),to determine whether there is a FA present and to establish itsidentity (Table 2). Both structures have PEG modeled in thetunnel. Both structures indeed have a bound FA that makessimilar interactions to those described above for the S. aureusFakBs. R. gnavus FakB (new PDB code 5V85) was successfullyrefined with cis-vaccenic acid (18:1�11), a major monounsatu-rated FA of E. coli, and E. eligens was refined with a saturatedFA, 17:0 (new PDB code 5UXY). These analyses suggest that allthe deposited FakB structures will turn out to have a FA, or amixture of FA, bound in their tunnels.

Comparison of the FakB1(16:0) and FakB2(18:1) structures

Despite the similarities in the overall structures of FakB1 andFakB2, the two proteins clearly possess different internal orga-

nizations that create FA binding tunnels with distinctly differ-ent shapes (Fig. 2, D and E). The most significant difference isthat, whereas the FakB1 tunnel is slightly curved, the FakB2(18:1�9) tunnel has a sharp turn at the 9-carbon of the bound FA toaccommodate the kink in the acyl chain that is present due tothe 9-cis conformation of the double bond. This significant dif-ference is accomplished by amino acid alterations at key loca-tions within the proteins’ hydrophobic cores to create thesespecific tunnel shapes. Most notably, the FakB1 residues Phe-193, Ile-198, and Phe-263 occupy the region where the hydro-carbon chain following the cis double bond is found in theFakB2 tunnel, whereas FakB2 residues Gly-273, Val-275, andLeu-155 occupy the space where the distal end of the saturatedFA tunnel in FakB1 is located. Ile-198 of FakB1 sits under the

Figure 2. FakB1 acyl chain binding tunnel. A, isolated 2Fo � Fc electron densities of the FakB1 FA contoured at the 1 � level. From top to bottom: teal � 14:0; green �a15:0; gray � 16:0; orange � a17:0. In the structures, the a17:0 is only observed in the naturally occurring S enantiomeric form, whereas the a15:0 fatty acid occurs inboth enantiomeric forms. B and C, orthogonal views of the FakB1 FA tunnel loaded with C14:0 (teal) and S-a17:0 (orange). The FA are represented as balls and sticks inthe middle of both images. C, black dotted line between the CD1 carbon atoms of the two conformations of Ile-233 shows how it moves 1.9 Å to accommodate thelonger a17:0 FA. In addition, the structural alignment reveals that Ala-158 and Leu-160 at the end of the tunnel are shifted by �0.5 Å to fit the longer a17:0 (orange)compared with 14:0 (teal). The FA-only alignment was performed with PSICO/MCSALIGN plugin for PyMOL 1.8 (Schrödinger, LLC). D and E, comparison of the distalends of the FA tunnels of FakB1(16:0) (PDB code 5UTO) (gray, D) and FakB2(18:1�9) (PDB code 4X9X) (blue, E). The FA are represented as balls and sticks. B–E, the meshesdelineate the cavities created within the protein to accommodate the FA and were computed with CAVER/PyMOL.

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FA in Fig. 2D at the position where the kink in the tunnel occursin FakB2. Whereas the kinked 18:1�9 chain cannot fit withinthe slightly curved hydrophobic tunnel of FakB1, the flexible16:0 chain should be able to fit into the kinked FakB2 tunnel.

FakB delivery of FA�P to PlsY and PlsX

We have been unable to isolate FakB proteins without abound FA. The FA remains bound unless there is a compatibleFA present in phospholipid vesicles or detergent micelles withwhich it can exchange (7). FakB that lacks a FA is unstable invitro and readily precipitates from solution. The importance ofthe interactions between the protein and the FA carboxyl arecritical for the thermal stability of FakB2 (7). The deletion ofone hydrogen bond in the FakB2(S93A) mutant lowers thedenaturation temperature from 50 to 33 °C (7), and the absenceof the FA would result in proteins even more unstable becausethe entire stabilizing hydrogen bond network would be elimi-nated. For example, FakB2 expression in Escherichia coli with18:1 in the medium results in high levels of protein expressionand the recovery of 100% soluble FakB2(18:1). The same exper-iment in the absence of 18:1 in the medium achieved the samelevel of protein expression, but it only results in 50% recovery ofsoluble FakB2 that contained 18:1 obtained from the E. coli host(data not shown). However, in vivo, FA-free FakB proteins mustexist because FakB(FA�P) is used as an acyl donor for at leasttwo downstream enzymes. To confirm this, we first determined

whether PlsY utilizes FA�P from FakB2(FA�P) (Fig. 3A).Assays containing FakA, FakB2(18:1), [14C]glycerol-phosphate,and membranes derived from S. aureus strain PDJ39 (�plsX)containing PlsY resulted in the rapid formation of LPA. Theformation of LPA was saturable, suggesting that the transfer ofFA�P occurred by FakB2 binding to PlsY (Fig. 3B). Althoughthese data are consistent with FA�P bound to FakB2 beingdirectly transferred to PlsY, one cannot completely rule outthat the FA�P bound to FakB2 was first deposited in themembrane where it subsequently encountered the acyltrans-ferase. To rule out this possibility, we next examined thetransfer of FA�P bound to FakB2 to a soluble protein part-ner, PlsX. We exchanged [14C]18:1 onto FakB2 and then iso-lated the FakB2([14C]18:1) complex by gel-filtration chro-matography to remove unbound [14C]18:1. Assays with PlsXand ACP led to the complete conversion of the 18:1�P fromFakB2 to acyl-ACP (Fig. 3C). Gel-filtration chromatographywas also used to demonstrate that all the FakB2([14C]18:1)was transferred to [14C]18:1-ACP in a FakA-dependent man-ner (Fig. 3D). FakA, PlsX, and ACP are all soluble proteins,and there was no detergent in the assay. These data meanthat FA-free FakB2 must be a product of the reaction andthat FA�P bound to FakB is removed from the proteinthrough its interaction with downstream metabolic enzymesleaving FA-free FakB.

Table 2Re-analysis of two PDB entries to identify bound fatty acidAvailable data collection statistics from the original PDB entries and refinement ofthe two PDB entries with heptadecanoic acid (17:0) and cis-vaccenic acid (18:1�11)are shown. NA means not applicable.

Fatty acids 17:0 18:1�11

PDB codes 5UXY(corrected 3FDJ)

5V85(corrected 3JR7)

Data collectionTemperature (K) 100 100Wavelength (Å) 0.97942–0.97929 0.9794Space group P 41 21 2 P 31 2 1Unit cell parameters (Å)

a, b, c 57.45, 57.45, 186.56 96.90, 96.90, 159.92�, �, � 90.00, 90.00, 90.00 90.00, 90.00, 120.00

Resolution range (Å) 48.91–1.80 83.92–2.00Rmerge 0.132 (0.804) (0.352)No. of unique reflections 30,039 (1990) 58,785 (4131)Multiplicity 7.7 (7.8)Mean I/�(I) 19.9 (2.0) 17.9 (2.1)Mean (I) half-set correlation

CC(1/2)NA NA

Completeness (%) 99.5 (97.2) 99.4 (99.5)Wilson B-factor (�2) 18.71 22.26

Model qualityRwork/Rfree value (%) 15.3/19.6 16.5/19.8Root mean square deviations 0.017 / 1.403 0.003/0.533

Bond lengths (Å) 0.18 0.19Bond angles (°) 1.35 0.54

Coordinates error (maximumlikelihood, Å)

0.18 0.19

Protein residues 276 557Average B-factor (Å2)

All atoms 24.21 30.45Protein atoms 21.90 28.75Fatty acid atoms 27.87 38.84Solvent atoms 37.14 41.79

Ramachandran plotFavored (%) 98.54 97.81Allowed (%) 1.46 2.19Outliers (%) 0.00 0.00

Clashscore 3.21 4.09

Figure 3. Transfer of FA�P from FakB to PlsY and PlsX. A, membranesprepared from strain PDJ39 (�plsX) were used as a source of PlsY. The panelshows the protein-dependent formation of acyl-[14C]glycerol-phosphate(LPA) in the presence of FakB2 and FA kinase illustrating that FakB2(18:1) isutilized by PlsY. B, dependence of PlsY activity on the FakB2 concentration inthe assay. FakB2([14C]18:1) was prepared by exchange with [14C]18:1, fol-lowed by the removal of unbound [14C]18:1 by gel-filtration chromatography.C, transfer of FakB2([14C]18:1) to ACP catalyzed by PlsX measured using thefilter disc assay to detect [14C]acyl-ACP formation. D, formation of [14C]18:1-ACP from PlsX and FakB2([14C]18:1). Assays contained FakB2([14C]18:1), ACPand PlsX. Samples were separated by gel-filtration chromatography on Sep-harose S-200 resin to illustrate the quantitative transfer of [14C]18:1 fromFakB2 to ACP. Details of the individual assays used in this figure are foundunder “Experimental procedures.”

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Status of the FakBs in vivo

We developed a cellular thermal shift assay (CETSA) toinvestigate whether FakB contains a bound FA in vivo.Ligand binding typically stabilizes proteins to thermal dena-turation, and the CETSA technique is commonly used indrug discovery to monitor drug-target engagement insidecells (12, 13). If FakB has a bound FA, it will be more stable tothermal denaturation than in the absence of FA (7). These invivo experiments required the generation of antibodies thatspecifically detect either FakB1 or FakB2. We generatedpolyclonal rabbit antibodies and purified the IgG fractionby affinity chromatography on FakB1- or FakB2-Sepharose.These affinity-purified antibodies only signaled the proteinof interest in immunoblots of whole S. aureus extracts (Fig.4). Anti-FakB1 did not cross-react with FakB2 (Fig. 4A), andanti-FakB2 did not cross-react with FakB1 (Fig. 4B). Webegan by analyzing FakB2 because this FA-binding proteinprefers 18:1, a FA that is abundant in mammalian hosts but isnot produced by S. aureus. The addition of 18:1 to the cul-ture medium resulted in a significant stabilization of FakB2to thermal denaturation (Fig. 5A). The results from threeindependent replicates showed that there was a shift of6.2 °C in the stability of FakB2 following exposure to 18:1.These data indicate that a significant proportion of FakB2does not contain a bound FA unless 18:1 was provided in themedium. We next performed the reverse experiment. Cells

were first grown with oleate, and a sample was obtained. Then,the 18:1 was removed from the medium; the cells were grownfor 1 h to deplete the cellular 18:1 by incorporation into phos-pholipid, and the second sample was harvested. The FakB2population was stabilized to heat denaturation in the presenceof 18:1 and returned to its less stable state following the removalof oleate from the medium (Fig. 5B). These data are consistentwith the majority of FakB2 existing in the cell without a boundligand, unless 18:1 was supplied in the medium.

FakB1 behaved differently. FakB1 had a high-thermal sta-bility in vivo, and there was no change in the FakB1 status

Figure 4. Validation of FakB1 and FakB2 antibody specificity. A, Coo-massie-stained and Western blotting of a series of isogenic S. aureusstrains (2) to validate the specificity of anti-FakB1. B, Coomassie-stainedand Western blotting of a series of isogenic S. aureus strains to validate thespecificity of anti-FakB2. Lane 1, purified protein; lane 2, strain AH1263(USA300); lane 3, strain JLB27 (�fakB1); lane 4, strain JLB28 (�fakB2); lane 5,strain JLB31 (�fakB1, �fakB2); lane 6, purified protein; lane 7, strainAH1263 (USA300); lane 8, strain JLB27 (�fakB1); lane 9, strain JLB28(�fakB2); lane 10, strain JLB31 (�fakB1, �fakB2). The purified FakBs areslightly higher molecular weight than the FakBs detected in vivo becausethey contain an N-terminal His6-tag.

Figure 5. CETSA. A, results of CETSA of FakB2 following a 1-h exposure ofS. aureus strain Sa178R1 to 500 �M exogenous 18:1. Western blottings as afunction of temperature for the two conditions are shown at the top of thepanel, and the densitometric trace is shown below. RT, room temperature. B,CETSA showing stabilized FakB2 becomes unstable when 18:1 is removedfrom the medium. Strain Sa178R1 cells were grown with 500 �M 18:1, then theculture was split, and 18:1 was removed from one culture. The cells wereharvested 1 h later and analyzed using the CETSA. C, CETSA of FakB1 in strainAH1263 before or after the addition of 500 �M exogenous 16:0. D, CETSA ofFakB2 before and after the addition of 500 �M extracellular 16:0. E, FakB1 wasoverexpressed in strain Sa178RI(pPJ464), and CETSA was performed on cellsgrown either in the absence and presence of 500 �M extracellular 16:0. Eachof these CETSA experiments was performed twice, and a representative resultis shown. Details of cell growth and the CETSA are found under “Experimentalprocedures.”

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when the cells were challenged with 16:0 (Fig. 5C). The lackof a FakB1 response to exogenous 16:0 suggests that a signif-icant proportion of the protein possessed a bound FA even inthe absence of an exogenous FA supplement. FakB2 was sta-bilized by 7.8 °C following a 16:0 challenge (Fig. 5D), consis-tent with the ability of FakB2 to bind both 18:1 and 16:0 (seebelow). These data indicated that FakB2 exists mostly in aligand-free state in vivo, whereas a high proportion of FakB1contains a bound FA suggesting a role for FakB1 in recyclingthe saturated FA produced by S. aureus into phospholipid(3). We next performed the same experiment using a plasmidsystem to increase the FakB1 cellular concentration. Afterthe addition of 16:0, the FakB1 thermal stability increased by4.5 °C (Fig. 5E). The stability of overexpressed FakB1 in thepresence of 16:0 (Fig. 5E) was equivalent to the stability ofnormal levels of FakB1 in the absence of a FA supplement(Fig. 5C). The stability of FakB1 was lower in the absence of16:0 in this experiment suggesting that with the higher levelsof FakB1 expression, a proportion of the FakB1 was ligand-free unless 16:0 was added to the medium.

Physiological roles of FakB1 and FakB2 in extracellular FAactivation

The physiological roles of FakB1 and FakB2 in exogenous FAactivation for phospholipid synthesis were examined by label-ing a series of mutant S. aureus strains for 30 min with anequimolar mixture of [D4]16:0 and 18:1, and measuring thesynthesis of phosphatidylglycerol (PG) molecular species fromthese exogenous FA precursors (Fig. 6). Strain AH1263 (WT)exhibited significant incorporation of both FAs into PG molec-ular species (Fig. 6A). [D4]16:0 was elongated to [D4]18:0 and[D4]20:0, whereas 18:1 was elongated to 20:1. Thus, both FAwere activated to FA�P and were either used immediately forphospholipid synthesis by PlsY or converted to acyl-ACP byPlsX and elongated by S. aureus FASII prior to being convertedagain to FA�P for incorporation into the 1-position by PlsY. Afew percent of [D4]16:0 was converted to acyl-ACP and used byPlsC to create the new 18:1/[D4]16:0 PG molecular species (Fig.6A, green peak). In strain JLB27 (�fakB1), the amount of[D4]16:0 incorporation was decreased and 18:1 incorporation

Figure 6. Effect of FakB deletions on the competitive uptake of 16:0 and 18:1 by S. aureus. S. aureus WT and mutant strains were grown to mid-logphase and labeled for 30 min with a mixture of [D4]16:0 and 18:1 (15 �M each) as described under “Experimental procedures.” Lipids were extracted, andthe PG molecular species were determined to quantify the utilization of exogenous fatty acids for phospholipid synthesis. Blue peaks in the spectradenote molecular species derived from [D4]16:0; the peaks derived from 18:1 are red; and the green peak contains both [D4]16:0 and 18:1. A, represen-tative PG molecular species produced by strain AH1263 (WT) labeled with the fatty acid mixture. B, representative spectrum of PG molecular speciesproduced by strain JLB27(�fakB1) labeled with the fatty acid mixture. C, representative spectrum of PG molecular species produced by strain JLB28(�fakB2) labeled with the fatty acid mixture. D, quantification of fatty acid uptake by a panel of fatty acid kinase knockout strains. In these calculations,[D4]16:0 incorporation is the sum of molecular species with [D4]16:0 [D4]18:0 [D4]20:0, and 18:1 incorporation corresponded to the sum ofmolecular species containing 18:1 20:1.

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increased, consistent with the preference of FakB2 for 18:1 cou-pled with its ability to also utilize 16:0 (Fig. 6, B and D). StrainJLB27 retained the ability to incorporate [D4]16:0 albeit atlower levels than in the WT strain. In strain JLB28 (�fakB2), theincorporation of 18:1 was severely compromised (Fig. 6, C andD). The JLB2 (�fakA) and JLB31 (�fakB1,�fakB2) doubleknockout strains did not incorporate exogenous FA into phos-pholipid (Fig. 6D), consistent with FA kinase as the only path-way for exogenous FA activation in S. aureus. FakB2 prefers

unsaturated FA (18:1), but can also utilize saturated FA (16:0),whereas FakB1 used saturated FA (16:0), but was severely com-promised in the activation of unsaturated FA (18:1). Theseresults confirm the physiological impact of the differing FakBsubstrate selectivities on the utilization of exogenous FA forphospholipid synthesis.

Biochemical FA kinase assays were used to evaluate the selec-tivities of the FA kinase system for the two FA in the absence ofdownstream metabolism (Fig. 7). FakB2 was able to phosphor-ylate both [14C]16:0 and [14C]18:1 with about equal efficiencyunder these assay conditions (Fig. 7A), but FakB1 was defectivein supporting the phosphorylation of [14C]18:1 (Fig. 7B). The invivo selectivity for FA type in Fig. 6 was more stringent thanobserved in these in vitro assays. However, in vivo, FakA maynot be the rate-determining step in phospholipid synthesisfrom exogenous FA. This point was addressed by performingexperiments as described in Fig. 6 using a 30 �M concentrationof a single FA (Fig. 7C). In this type of experiment, there was nodifference between the uptake of [D4]16:0 and 18:1 by strainJLB27 (�fakB1) (Fig. 7C), consistent with the biochemistryexperiment (Fig. 7, A and B). In strain JLB28 (�fakB2), 18:1incorporation was severely impaired (Fig. 7C), although theamount of 16:0 incorporated in this knockout strain remainedrobust. These experiments confirmed that unsaturated FA areprevented from interacting with the substrate binding tunnel ofFakB1. FakB2 prefers to bind unsaturated FA but did notexclude the flexible saturated FA that can assume the shape ofthe kinked tunnel.

Discussion

Our results lead to a model for the function of FakBs inS. aureus physiology diagrammed in Fig. 8. FakB1 bindsstraight, and branched-chain saturated FA that are produced bythe S. aureus FA synthase. In the absence of FA kinase activity,the intracellular FA pool rises indicating that one housekeepingfunction of the kinase is to activate these FA so they can berecycled into phospholipid (3). The metabolic origin of theS. aureus FA pool remains unknown, but they are all saturatedFA, and FakB1 has the substrate selectivity consistent with a

Figure 7. FA activation by FakB1 or FakB2. FA kinase assays were per-formed to measure the formation of acyl-PO4 from [14C]16:0 or [14C]18:1 (20�M) as a function of FakB1 or FakB2 concentration. A, rate of 16:0-PO4 forma-tion as a function of either the FakB1 or FakB2 concentration. B, rate of 18:1-PO4 formation as a function of either the FakB1 or FakB2 concentration. C,S. aureus strains AH1263, JLB27 (�fakB1), or JLB28 (�fakB2) were labeled with30 �M of either [D4]16:0 or 18:1 for 30 min. The cells were harvested andextracted, and the amount of each FA incorporated into phospholipid wasdetermined by MS. Details of the FA kinase assays, metabolic labeling, and MSare found under “Experimental procedures.”

Figure 8. Roles of FakB1 and FakB2 in S. aureus physiology. ExogenousFA, either saturated (SFA) or monounsaturated (18:1), are incorporated intomembrane phospholipids following activation by FA kinase. SFA are boundto FakB1 and FakB2 prefers 18:1, although it can bind SFA as indicated by thedotted line also. Both fatty acid-binding proteins are substrates for FakA-me-diated phosphorylation. The acyl-phosphates bound to the FakBs are thentransferred to either PlsY (sn-glycero-3-phosphate (G3P) acyltransferase) toform acyl-G3P (LPA) to initiate membrane phospholipid (PL) synthesis, or toPlsX, which converts the acyl-phosphate to acyl-ACP. Acyl-ACP enters theelongation cycle of fatty acid biosynthesis (FASII). The origin of cellular FA inS. aureus is unknown, but CETSA data suggest FakB1 functions to bind thesesaturated FA for activation by FakA and re-cycling into the PL biosyntheticpathway. FakB2 can substitute for FakB1, but its unique function is to activateextracellular host 18:1 to be utilized for phospholipid lipid synthesis at theinfection site.

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role in activating these FA. The CETSA results suggest that asignificant proportion of FakB1 may be bound to FA duringS. aureus growth, consistent with a role for FakB1 in the re-cy-cling of saturated FA to maintain lipid homeostasis. S. aureusdoes not synthesize unsaturated FA, and the CETSA resultsindicate that a significant proportion of FakB2 is not bound to aFA unless 18:1 is supplied in the medium. Oleate (18:1) is anabundant mammalian FA that is present at S. aureus infectionsites. FA synthesis is an energy-intensive process, and theexpression of the two FA-binding proteins enables S. aureus toutilize both saturated and unsaturated host resources for theformation of phospholipids during growth at the infection site.

This work shows that distinct differences within the hydro-phobic protein cores of bacterial FA-binding proteins accountfor the FA acyl chain selectivity characteristic of the FakB pro-teins. FakB1 binds a series of saturated and branched-chain FAthat are produced by S. aureus within a linear hydrophobic tun-nel buried within the FakB1 core. Ile-233 at the end of the bind-ing tunnel acts as a flexible plug that repositions to allow bind-ing of 14 –17 carbon chain lengths. Unsaturated FA have akinked structure due to the cis double bond, making it a poor fitfor the FakB1 tunnel. This accounts for the low rate of FakB1-dependent FA kinase activity in vitro when oleate is the sub-strate and for the compromised ability of strains that onlyexpress FakB1 to incorporate exogenous 18:1 into phospho-lipid. In contrast, the FakB2 tunnel is almost an exact match forthe conformation of 18:1. This tunnel architecture prefers tobind 18:1, but it does not exclude saturated FA because theseFA are flexible enough to negotiate the kinked tunnel. Cellsexpressing only FakB2 incorporate both saturated and mono-unsaturated FA, although FakB2 shows a clear preference for18:1 when presented in a FA mixture. We conclude that FakB1is the primary housekeeping FA-binding protein responsiblefor activating cellular saturated FA that arise from lipidmetabolism, and FakB2 functions to activate and exploitunsaturated FA that are available at the infection site (Fig. 8).The existence of FA-specific binding pockets is a feature ofbacterial FakBs that is not shared by the mammalian FA-binding proteins, which bind a range of FA chain lengths andunsaturation (14, 15).

Although the bacterial FA-binding protein family is charac-terized by diverse hydrophobic protein interiors that accom-modate specific FA structures, these variations within the FakBinteriors do not extend to the FakB exterior surface. These sur-face features are critical for protein–protein interactions withFakB-binding partners. All FakB homologs interact with a sin-gle FakA kinase domain protein, and a conserved arginine (Arg-205 in FakB1 and Arg-202 in FakB2) has a key role at the dock-ing site (7). In addition, the FakB(FA�P) can donate FA�P toeither PlsX or PlsY illustrating that FakBs have at least twoother conserved protein partners in addition to FakA. Somebacteria express three (Streptococcus pneumoniae) or four(Enterococcus faecalis) FakBs, and it is likely that each of theseindividual proteins possesses a different organization of inte-rior hydrophobic residues to create tunnels that selectively binda specific subset of FA structures.

Experimental procedures

Bacterial strains and reagents

The bacterial strains and their origins are listed in Table 3.The FakB1 expression plasmid was constructed by ordering aDNA string from Invitrogen based on the fakB1 sequence ofstrain AH1263 that was cloned into BamHI and XbaI sites ofplasmid pG164 to yield pPJ464 and sequenced. The expressionplasmid was transformed into strain Sa178RI by electropo-ration and selected on chloramphenicol plates. [14C]16:0(specific activity, 55 mCi/mol) and [14C]18:1 (specific activ-ity, 59 mCi/mol) were purchased from PerkinElmer Life Sci-ences. [14C]Glycerol-phosphate (specific activity, 50 mCi/mol) was purchased from American Radiochemicals. FAwere purchased from Sigma, and 7,7,8,8- tetradeuteriohexa-decanoic acid ([D4]16:0) was obtained from Cambridge Iso-tope Laboratories, Inc.

Protein crystallization and structure determination

Crystals of FakB1(FA), all in space group P1 with very similarcell dimensions, were obtained by mixing equivalent volumes ina sitting drop of protein solution at 10 –20 mg/ml concentra-tion (100 mM NaCl, 10 mM Tris, pH 7.5) with the precipitantsolution. The optimized crystallization conditions (MorpheusC4, Molecular Dimensions) were as follows: 0.1 M MES, imid-azole, pH 6.5, 0.03 M sodium nitrate, 0.03 M disodium hydrogenphosphate, 0.03 M ammonium sulfate, and 12.5% PEG 1000,12.5% PEG 3350, 12.5% MPD. Crystals typically appearedwithin 5–7 days. The crystals were cryoprotected with the pre-cipitant mother liquor containing 12.5% glycerol and frozen inliquid nitrogen prior to data collection. X-ray diffraction data(360°) were collected on single crystals at SER-CAT beamlineID22 and BM22 at APS Argonne. Diffraction data were inte-grated with XDS (16, 17) and scaled using AIMLESS/CCP4(18) to 1.63–1.93 Å resolution depending on the sample. TheFakB1(16:0) structure was first determined by molecularreplacement using a polyalanine version of the analog PDBdatabase model 4X9X (FakB2) as the search model usingPHASER-MR/CCP4 (19). The final refined model of FakB1contained two monomers per asymmetric unit, each containing16:0 (Table 1). Subsequent FakB1 structures were determinedby molecular replacement with MOLREP (20) using this initialstructure (Table 1). For the re-refinement of PDB structures3JR7 and 3FDJ, the PEG molecules originally modeled in theFakB tunnels were replaced by fatty acid molecules, and thestructure factors (.cif files) were obtained from the PDB data-

Table 3IPTG is isopropyl 1-thio-�-D-galactopyranoside

Description Ref.

StrainAH1263 USA300 Nebraska collection 27JLB2 �fakA of strain AH1263 6JLB27 fakB1::�� of strain AH1263 2JLB28 fakB2::�� of strain AH1263 27JLB31 fakB1::�� �fakB2 of strain AH1263 2Sa178RI Derived from RN4220 28PDJ39 �plsX of strain Sa178RI 1

PlasmidspG164 IPTG-inducible vector 28pPJ464 IPTG-inducible FakB1 expression This study

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base and converted to .mtz file using IMPORT/CCP4i (Table 2)(21). In all cases, the 3D coordinates for the FA molecules weredesigned using AVOGADRO (22), and the .cif dictionaries ofrestraints were generated with PHENIX.ELBOW (23). Manualand real space refinements were performed with COOT andREFMAC, respectively, and water molecules were placed bothautomatically and manually with COOT (24). In all refine-ments, 5% of the reflections were excluded for calculation of theRfree values.

Expression, purification, and FA exchange

FakA, FakB1, and FakB2 were purified as described previ-ously (2). Purified FakB1 was loaded with a mixture of saturatedFA found in the E. coli expression system (mostly 16:0). FakB1with specific FA bound were prepared as described (7). Briefly,FA were added during cell lysis before protein purification, andthe protein isolated after Ni2-affinity purification was sub-jected to gel-filtration chromatography and concentrated forcrystallization studies. S. aureus PlsX was purified as describedin Ref. 1. PlsY membranes were purified as described in Ref. 25.Briefly, strain PDJ39 (�plsX) was grown to an A600 of 2.0 andlysed with 5 mg/ml lysostaphin in 20 mM potassium phosphatebuffer, pH 7.5, 5 mM EDTA, 50 mM KCl, 10 mM MgCl2 withprotease inhibitor mixture at 37 °C for 1 h. Cells were furtherdisrupted with a French press, and the cell debris was removedby centrifugation at 10,000 � g for 10 min. Membranes wereisolated by centrifuging the supernatant at 200,000 � g for 45min, and the membrane pellet was resuspended in 20 mM

potassium phosphate buffer, pH 7.5, 50 mM KCl, 10 mM MgCl2and used for the experiment.

PlsX and PlsY assays

To study the interaction of FakB2 and PlsX, FA kinase assaywith 0.5 �M FakA and 5 �M FakB2 exchanged with [14C]18:1was added to a reaction mix containing 100 mM Tris, pH 7.5, 20mM MgCl2, 10 mM ATP and incubated at 37 °C for 30 min. Tothis either 25 �M PlsX or 25 �M PlsX and 250 �M S. aureus ACPwas added in a final reaction volume of 50 �l and incubated at37 °C for 20 min. Aliquots (40 �l) were spotted on Whatman3MM disks and washed twice with chloroform/methanol/ace-tic acid (3:6:1, v/v) mixture (20 ml per disk) for 20 min for eachwash. The disks were dried and counted by scintillation count-ing. The experiments were performed twice in duplicate.

To study the interaction of FakB2 and PlsY, FA kinase assayswith 0.5 �M FakA and 20 �M FakB2 was added to a reaction mixcontaining 100 mM Tris, pH 7.5, 20 mM MgCl2, 10 mM ATP andincubated at 37 °C for 30 min. This was added to a mixturecontaining 150 mM NaCl, 100 �M [14C]glycerol-phosphate, 1mg/ml BSA, 5 mM Na3VO4, and membranes prepared fromstrain PDJ39 (�plsX) and incubated for 20 min at 37 °C. A 40-�laliquot was spotted on Whatman 3MM disks and washed with10%, 5%, and 1% TCA (20 ml per disk) for 20 min for each wash.The disks were dried and counted by scintillation counting. Theexperiments were performed twice in duplicate.

Gel filtration

To test the transfer of fatty acid from FakB2 to ACP via PlsX,10 �M FakB2 exchanged with [14C]18:1 was added to a reaction

mix containing 100 mM Tris, pH 7.5, 20 mM MgCl2, 10 mM ATPwith or without 0.5 �M FakA and incubated at 37 °C for 30 min.To this, 25 �M PlsX and 250 �M ACP was added to a finalreaction volume of 100 �l and incubated at 37 °C for 20 min.This was loaded onto a Sephadex S-75 column and eluted with20 mM Tris, pH 7.5, 200 mM NaCl. 200-�l fractions were col-lected, and 100 �l was counted by scintillation counting. Theexperiment was performed twice.

Antibody generation and testing

The antibodies for FakB1 and FakB2 were generated inrabbit against purified His-tagged FakB1 and FakB2 proteinsby Rockland Antibodies Inc. The specificity of the FakB1 andFakB2 antibodies was analyzed using extracts from strainsAH1263 (WT USA300), JLB27 (�fakB1), JLB28 (�fakB2),and JLB31 (�fakB1 and �fakB2). Lysates were resolved usinga 10% bis-tris acrylamide SDS gel and transferred to a poly-vinylidene difluoride membrane. The blots were blocked for1 h in 1% milk/TBS-T and then exposed to primary antibody(FakB1 or FakB2 as indicated in Fig. 4) overnight at 1:1000dilution in 1% BSA/TBS-T followed by secondary antibody(anti-rabbit AP-conjugated, GE Healthcare ECF Westernblotting reagent pack) in 1% milk/TBS-T for 1 h at 1:5000dilution. The blot was washed extensively and exposed to theECF substrate for 5 min, and the bands on the dried mem-brane were quantified on the Typhoon FLA9500 using Image-Quant TL software (GE Healthcare).

Cellular thermal shift assay (CETSA)

Strains Sa178RI or AH1263 were grown to an A600 of 0.5 inLuria broth containing 10 mg/ml FA-free bovine serum albu-min (BSA) and treated with either DMSO or 500 �M FA (18:1 or16:0) in DMSO as indicated in Fig. 5 for 40 min. Cells wereharvested and resuspended in resuspension buffer (20 mM Tris,pH 8.0, 200 mM NaCl, and protease inhibitors) such that theA600 is 1.0 in the buffer. Aliquots (100 �l) in 0.5-ml reactiontubes were exposed to various temperatures (room tempera-ture and 42, 45, 48, 51, 54, 57, 60, and 63 °C) in pairs (controland FA-treated) using a thermocycler PCR machine for 3 min,then moved to room temperature for another 3 min beforebeing flash-frozen in liquid nitrogen, and stored at �80 °C. Forthe FA–pre-loaded experiments, cells were inoculated at anA600 of 0.05, and 500 �M FA was added to the media containing10 mg/ml FA-free BSA. Cells were grown to an A600 of 0.5, theneither washed with media containing 10 mg/ml FA-free BSAor not washed and grown further for two doublings. Growingcells were harvested and processed as described above. StrainSa178RI/pPJ464 was grown; the expression of FakB1 wasinduced with 200 �M isopropyl 1-thio-�-D-galactopyranosidein the medium, and the cells were grown with FA or DMSO andprocessed as described above. The frozen cell samples fromeach thermal denaturation point were lysed by adding 2 �l oflysostaphin (5 mg/ml) followed by incubation on ice for 15 minand then sonicated at 4 °C for 10 s. The lysates were centrifugedat 14,000 � g for 20 min to remove precipitated proteins.Immunoblotting was performed, and the bands were quantifiedas described above. All the experiments were repeated twice,

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and the data shown is a representative graph from one of theblots.

FA incorporation

Overnight cultures were used to inoculate 5 ml of fresh Luriabroth (LB) plus 0.1% Brij58 to an A600 of 0.04. Cultures were incu-bated at 37 °C to an A600 of 0.5 and were then split into 0.2% DMSO(control), 30 �M [D4]16:0 or -18:1 individually, or a mix of 15 �M

[D4]16:0 15 �M 18:1 and incubated at 37 °C for 30 min. Cellswere harvested and washed twice with LB and once with PBS, andlipids were extracted using the Bligh and Dyer method (26).

Lipid extracts were resuspended in chloroform/methanol(1:1). Phosphatidylglycerol (PG) was analyzed using a Shi-madzu Prominence UFLC attached to a QTrap 4500 equippedwith a Turbo V ion source (Sciex). Samples were injected ontoan Acquity UPLC BEH HILIC, 1.7 �M, 2.1 � 150-mm column(Waters) at 40 °C and a flow rate of 0.2 ml/min. Solvent A wasacetonitrile, and solvent B is 15 mM ammonium formate, pH 3.The HPLC program was as follows: starting solvent mixture of96% A, 4% B, 0 –2 min isocratic with 4% B; 2–20 min lineargradient with 80% B; 20 –23 min isocratic with 80% B; 23–25min linear gradient with 4% B; 25–30 min isocratic with 4% B.The QTrap 4500 was operated in the negative mode, and theion source parameters were s follows: ion spray voltage, �4500V; curtain gas, 25 p.s.i.; temperature, 350 °C; collision gas,medium; ion source gas 1, 40 p.s.i.; ion source gas 2, 60 p.s.i.;and declustering potential, �40 V. The system was con-trolled by the Analyst� software (Sciex), and LipidViewTM

software (Sciex) was used to analyze and quantitate the PGmolecular species.

The percentages of each peak in the mass spectra wereobtained by calculating the area under each peak and then cal-culating the percent of the total molecular species that weresynthesized from exogenous FA. [D4]16:0 uptake was deter-mined by summing the peaks containing [D4]16:0, [D4]18:0, or[D4]20:0, and 18:1 incorporation were peaks containing either18:1 or 20:1. The small peak containing both [D4]16:0 and -18:1was counted as incorporation for both FA.

Fatty acid kinase assay

FA kinase assays were performed with 0.2 �M FakA and 20�M [14C]16:0 or [14C]18:1, 100 �M Tris, pH 7.5, 20 mM MgCl2,10 mM ATP, and 1% Triton X-100. After reactions were assem-bled, FakB1 or FakB2 was added at the indicated concentrationsto initiate the reaction and incubated at 37 °C for 20 min. Analiquot (40 �l) of the reaction mixture was spotted onto DE81Whatman filter paper discs. Discs were washed three timeswith ethanol, 1% acetic acid mixture for 20 min for each wash.Discs were then dried and counted by scintillation counting.Experiments were each performed two times in duplicate.

Author contributions—M. G. C., C. S., M. W. F., and S. W. W. for-mal analysis; M. G. C. validation; M. G. C., C. S., J. M. G., M. W. F.,and S. W. W. writing-review and editing; C. S. and C. O. R. concep-tualization; C. S. and S. W. W. data curation; C. S., J. M. G., andM. W. F. investigation; C. S., S. W. W., and C. O. R. project adminis-tration; S. W. W. and C. O. R. supervision; C. O. R. funding acquisi-tion; C. O. R. writing-original draft.

Acknowledgments—We thank Tyler Broussard for obtaining theinitial crystals of FakB1(16:0); Megan Ericson for input at the begin-ning of the project; Caroline Pate for CETSA experiments; Katie Wellsfor biochemical assays; and Karen Miller for protein purificationand FA analysis. The X-ray diffraction data were collected at APSbeamline SER-CAT ID-22 and BM-22. Use of the Advanced PhotonSource was supported by the United States Department of Energy,Office of Science, Office of Basic Energy Sciences, under Contract No.W-31-109-Eng-38.

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Stephen W. White and Charles O. RockMaxime G. Cuypers, Chitra Subramanian, Jessica M. Gullett, Matthew W. Frank,

binding proteins− fatty acidStaphylococcus aureusAcyl-chain selectivity and physiological roles of

doi: 10.1074/jbc.RA118.006160 originally published online November 14, 20182019, 294:38-49.J. Biol. Chem. 

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